It has become increasingly clear in recent years that cell death is as important to the health of a multicellular organism as cell division: where proliferation exists, so must a means of regulating its cellular progeny. By repeated cell division and differentiation throughout development or tissue repair, surplus or even harmful cells are generated, and they must be removed or killed. In adults, senescent cells are removed and replaced by newly generated cells to maintain homeostasis.
The delicate interplay between growth and cell death in an organism is mirrored in the complex molecular balance that determines whether an individual cell undergoes division; arrests in the cell cycle; or commits to programmed cell death. Signal transduction is the term describing the process of conversion of extracellular signals, such as hormones, growth factors, neurotransmitters, cytokines, and others, to a specific intracellular response such as gene expression, cell division, or apoptosis. This process begins at the cell membrane where an external stimulus initiates a cascade of enzymatic reactions inside the cell that typically include phosphorylation of proteins as mediators of downstream processes which most often end in an event in the cell nucleus. The checks and balances of these signal transduction pathways can be thought of as overlapping networks of interacting molecules that control xe2x80x9cgo-no goxe2x80x9d control points. Since almost all known diseases exhibit dysfunctional aspects in these networks, there has been a great deal of enthusiasm for research that provides targets and therapeutic agents based on signal transduction components linked to disease.
Dysregulation of cell proliferation, or a lack of appropriate cell death, has wide ranging clinical implications. A number of diseases associated with such dysregulation involve hyperproliferation, inflammation, tissue remodelling and repair. Familiar indications in this category include cancers, restenosis, neointimal hyperplasia, angiogenesis, endometriosis, lymphoproliferative disorders, graft-rejection, polyposis, loss of neural function in the case of tissue remodelling, and the like. Such cells may lose the normal regulatory control of cell division, and may also fail to undergo appropriate cell death.
In one example, epithelial cells, endothelial cells, muscle cells, and others undergo apoptosis when they lose contact with extracellular matrix, or bind through an inappropriate integrin. This phenomenon, which has been termed xe2x80x9canoikisxe2x80x9d (the Greek word for xe2x80x9chomelessnessxe2x80x9d), prevents shed epithelial cells from colonizing elsewhere, thus protecting against neoplasia, endbmetriosis, and the like. It is also an important mechanism in the initial cavitation step of embryonic development, in mammary gland involution, and has been exploited to prevent tumor angiogenesis. Epithelial cells may become resistant to anoikis through overactivation of integrin signaling. Anoikis resistance can also arise from the loss of apoptotic signaling, for example, by overexpression of Bcl-2 or inhibition of caspase activity.
An aspect of hyperproliferation that is often linked to tumor growth is angiogenesis. The growth of new blood vessels is essential for the later stages of solid tumor growth. Angiogenesis is caused by the migration and proliferation of the endothelial cells that form blood vessels.
In another example, a major group of systemic autoimmune diseases is associated with abnormal lymphoproliferation, as a result of defects in the termination of lymphocyte activation and growth. Often such diseases are associated with inflammation, for example with rheumatoid arthritis, insulin dependent diabetes mellitus, multiple sclerosis, systemic lupus erythematosus, and the like. Recent progress has been made in understanding the causes and consequences of these abnormalities. At the molecular level, multiple defects may occur, which result in a failure to set up a functional apoptotic machinery.
The development of compounds that inhibit hyperproliferative diseases, particularly where undesirable cells are selectively targeted, is of great medical and commercial interest.
Relevant Literature
Triazolylated tertiary amine compounds are provided in U.S. Pat. Nos. 5,674,886. 4,101,548 and 4,171,363 disclose quinazoline compounds, and in particular 2-piperazinyl-6,7-dimethoxyquinazolines compounds that include a 1,2,3-thiadiazole terminal group. U.S. Pat. Nos. 3,787,434 and 3,874,873 disclose herbicidal compounds and compositions that include 1,2,3-thiadiazole-5-yl ureas.
Chemical compounds are disclosed in Tarasov et al., Khim. Geterotsikl. Soedin. 8:1124-1127, 1996; Morzherin, Tarasov and Bakulev, Khim. Geterotsikl Soedin. 4:554-559, 1994; Morzherin, Bakulev, Dankova and Mokrushin, Khim. Geterotsikl Soedin 4:548-553, 1994; Shafran, Bakulev, Shevirin, and Kolobov, Khim. Geterotsikl. Soedin. 6:840-6, 1993; Dankova, Bakulev, and Morzherin, Khim. Geterotsikl. Soedin. 8:1106-1112, 1992; Bakulev, Lebedev, Dankova, Mokrushin, and Petrosyan, Tetrahedron 45(23):7329-7340, 1989; Kankova, Bakulev, Kolobov, Andosova, and Mokrushin, Khim. Geterotsikl, Soedin. 6:827-829, 1989; Dankova, Bakulev, Kolobov, Shishkina, Yasman, and Lebedev, Khim. Geterotsikl. Soedin 9:1269-1273, 1988; Bakulev, Kolobov, Grishakov, and Mokrushin, Izv. Akad. Nauk SSR, Ser. Khim. 1:193-195, 1988; Kolobov, Bakulev, Mokrushin, and Lebedev, Khim. Geterotsikl. Soedin 11:1503-1508, 1987; Bakulev, Dankova, Mokrushin, Sidorov, and Lebedev, Khim. Geterotsikl. Soedin. 6:845-849, 1987; Kolobov, Bakulev, and Mokrushin, Zh. Org. Khim. 23(5):1120-1122, 1987; Lebedev, Shevchenko, Kazaryan, Bakulev, Shafran, Kolobov, and Prosyan, Khim. Geterotsikl. Soedin. 5:681-689, 1987; Shafran, Bakulev, Mokrushin, and Validuda, Khim. Geterotsikl. Soedin. 5:691-696, 1986; Dankova, Bakulev, Mokrushin, and Shafran, Khim. Geterotsikl. Soedin, 10:1429-1430, 1985; and Shafran, Bakulev, Mokrushin, and Pushkareva, Khim. Geterotsikl. Soedin. 12:1696-1697, 1982; and Gewald and Hain, J. Prakt. Chem. 317(2):329-336,1975.
The regulation of integrin linked kinase by phosphatidylinositol (3,4,5) trisphosphate is described by Delcommenne et al. (1998) Proc Natl Acad Sci 95:11211-6. Activated nitriles in heterocyclic synthesis are discussed in Kandeel et al. (1985) J. Chem. Soc. Perkin. Trans 1499. Oxidative transformation of pyrazole into triazole is discussed in Kandeel et al. (1986) J. Chem. Soc. Perkin. Trans 1379.
Pharmaceutical compositions and compounds are provided. The compounds of the invention are 1,2,3 thiadiazole compounds. In one embodiment of the invention, formulations of the compounds in combination with a physiologically acceptable carrier are provided. The pharmaceutical formulations are useful in the treatment of disorders associated with hyperproliferation and tissue remodelling or repair. The compounds are also active in the inhibition of specific protein kinases.
The present invention provides novel compounds, compositions and methods as set forth within this specification. In general, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs, unless clearly indicated otherwise. For clarification, listed below are definitions for certain terms used herein to describe the present invention. These definitions apply to the terms as they are used throughout this specification, unless otherwise clearly indicated.
As used herein the singular forms xe2x80x9caxe2x80x9d, xe2x80x9candxe2x80x9d, and xe2x80x9cthexe2x80x9d include plural referents unless the context clearly dictates otherwise. For example, xe2x80x9ca compoundxe2x80x9d refers to one or more of such compounds, while xe2x80x9cthe enzymexe2x80x9d includes a particular enzyme as well as other family members and equivalents thereof as known to those skilled in the art.
xe2x80x9cAcylxe2x80x9d is a specie of heteroalkyl wherein a terminal carbon of the heteroalkyl group is in the form of a carbonyl group, i.e., (alkyl or heteroalkyl)-Cxe2x95x90O, where examples include acetyl (CH3xe2x80x94(Cxe2x95x90O)xe2x80x94).
xe2x80x9cAcyloxyxe2x80x9d refers to a heteroalkylene group of the formula xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94 bonded to xe2x80x9cXxe2x80x9d so as to form xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94X wherein X may be any of alkyl, aryl, heteroalkyl, or heteroaryl.
xe2x80x9cAlkenylxe2x80x9d is a specie of alkyl group, where an alkenyl group has at least one carbon-carbon double bond.
xe2x80x9cAlkenylenexe2x80x9d is a specie of alkylene group where the alkylene group has at least one double bond.
xe2x80x9cAlkylxe2x80x9d is a monovalent, saturated or unsaturated, straight, branched or cyclic, aliphatic (i.e., not aromatic) hydrocarbon group. In various embodiments, the alkyl group has 1-20 carbon atoms, i.e., is a C1-C20 (or C1-C20) group, or is a C1-C18 group, a C1-C12 group, a C1-C6 group, or a C1-C4 group. Independently, in various embodiments, the alkyl group: has zero branches (i.e., is a straight chain), one branch, two branches, or more than two branches; is saturated; is unsaturated (where an unsaturated alkyl group may have one double bond, two double bonds, more than two double bonds, and/or one triple bond, two triple bonds, or more than three triple bonds); is, or includes, a cyclic structure; is acyclic. Exemplary alkyl groups include C1 alkyl (i.e., xe2x80x94CH3 (methyl)), C2 alkyl (i.e., xe2x80x94CH2CH3 (ethyl), xe2x80x94CHxe2x95x90CH2 (ethenyl) and xe2x80x94Cxe2x89xa1CH (ethynyl)) and C3 alkyl (i.e., xe2x80x94CH2CH2CH3 (n-propyl), xe2x80x94CH(CH3)2 (i-propyl), xe2x80x94CHxe2x95x90CHxe2x80x94CH3 (1-propenyl), xe2x80x94Cxe2x89xa1xe2x80x94Cxe2x95x90CH3 (1-propynyl), xe2x80x94CH2CHxe2x80x94CH2 (2-propenyl), xe2x80x94CH2xe2x80x94Cxe2x89xa1CH (2-propynyl), xe2x80x94C(CH3)xe2x95x90CH2 (1-methylethenyl), and xe2x80x94CH(CH2)2 (cyclopropyl)).
xe2x80x9cAlkylenexe2x80x9d is a polyvalent, saturated or unsaturated, straight, branched or cyclic, aliphatic (i.e., not aromatic) hydrocarbon group. In various embodiments, the alkylene group has 1-20 carbon atoms, i.e., is a C1-C20 group, or is a C1-C18 group, a C1-C12 group, a C1-C6 group, or a C1-C4 group. Independently, in various embodiments, the alkylene group: has zero branches (ie., is a straight chain), one branch, two branches, or more than two branches; is saturated; is unsaturated (where an unsaturated alkylene group may have one double bond, two double bonds, more than two double bonds, and/or one triple bond, two triple bonds, or more than three triple bonds); is or contains a cyclic group; is acyclic; is divalent, i.e., has two open sites that each bond to a non-alkylene group; is trivalent, i.e., has three open sites that each bond to a non-alkylene group; has more than three open sites. Exemplary alkylene groups include C1alkylene (i.e., xe2x80x94CH2xe2x80x94) and C2 alkylene (i.e., xe2x80x94CH2CH2xe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94C Cxe2x80x94, xe2x80x94C(xe2x95x90CH2)xe2x80x94, and xe2x80x94CH(CH3)xe2x80x94).
xe2x80x9cAralkenylxe2x80x9d is another name for arylalkenylene, wherein at least one of the open bonding sites of an alkenylene group is bonded to an aryl group.
xe2x80x9cAralkylxe2x80x9d is another name for arylalkylene, wherein at least one of the open bonding sites of an alkylene group is bonded to an aryl group, where benzyl is an example.
xe2x80x9cArylxe2x80x9d is a monovalent, aromatic, hydrocarbon, ring system. The ring system may be monocyclic or fused polycyclic (e.g., bicyclic, tricyclic, etc.). In various embodiments, the monocyclic aryl ring is C5-C10, or C5-C7, or C5-C6, where these carbon numbers refer to the number of carbon atoms that form the ring system. A C6 ring system, i.e., a phenyl ring, is a preferred aryl group. In various embodiments, the polycyclic ring is a bicyclic aryl group, where preferred bicyclic aryl groups are C8-C12, or C9-C10. A naphthyl ring, which has 10 carbon atoms, is a preferred polycyclic aryl group.
xe2x80x9cArylenexe2x80x9d is a polyvalent, aromatic hydrocarbon, ring system. The ring system may be monocyclic or fused polycyclic (e.g., bicyclic, tricyclic, etc.). In various embodiments, the monocyclic arylene group is C5-C10, or C5-C7, or C5-C6, where these carbon numbers refer to the number of carbon atoms that form the ring system. A C6 ring system, i.e., a phenylene ring, is a preferred aryl group. In various embodiments, the polycyclic ring is a bicyclic arylene group, where preferred bicyclic arylene groups are C8-C12, or C9-C10. A naphthylene ring, which has 10 carbon atoms, is a preferred polycyclic aryl group. The arylene group may be divalent, i.e., it has two open sites that each bond to another group; or trivalent, i.e., it has three open sites that each bond to another group; or it may have more than three open sites.
xe2x80x9cCycloalkenylxe2x80x9d is a specie of alkyl group where a cycloalkenyl group is a cyclic hydrocarbon group with at least one double bond.
xe2x80x9cCycloalkenylenexe2x80x9d is a specie of alkylene group which is a cyclic hydrocarbon with at least one double bond and with at least two bonding sites.
xe2x80x9cCycloalkylxe2x80x9d is a specie of alkyl group, where a cycloalkyl is a cyclic hydrocarbon group.
xe2x80x9cCycloalkylalkylenexe2x80x9d is a species of alkyl group wherein at least one open bonding site of an alkylene group is joined to a cycloalkyl group.
xe2x80x9cCycloalkylenexe2x80x9d is a specie of alkylene group which is a cyclic hydrocarbon group with at least two open bonding sites.
xe2x80x9cCycloalkylenealkylenexe2x80x9d is a specie of alkylene group wherein a cycloalkylene group is bonded to a non-cyclic alkylene group, and each of the cycloalkylene and non-cyclic alkylene group have at least one open bonding site.
Haloalkyl is a specie of heteroalkyl wherein at least one carbon of an alkyl group is bonded to at least one halogen.
xe2x80x9cHalogenxe2x80x9d refers to fluorine, chlorine, bromine and iodide. Fluorine and chlorine are preferred halogens in compounds and compositions of the present invention.
Heteroalkylenearyl is a heteroalkylene group with at least one of its open bonding sites joined to an aryl group, where benzoyl (xe2x80x94C(xe2x95x90O)xe2x80x94Ph) is an example.
xe2x80x9cHeteroalkylxe2x80x9d is an alkyl group (as defined herein) wherein at least one of the carbon atoms is replaced with a heteroatom. Preferred heteroatoms are nitrogen, oxygen, sulfur, and halogen. A heteroatom may, but typically does not, have the same number of valence sites as carbon. Accordingly, when a carbon is replaced with a heteroatom, the number of hydrogens bonded to the heteroatom may need to be increased or decreased to match the number of valence sites of the heteroatom. For instance, if carbon (valence of four) is replaced with nitrogen (valence of three), then one of the hydrogens formerly attached to the replaced carbon must be deleted. Likewise, if carbon is replaced with halogen (valence of one), then three (i.e., all) of the hydrogens formerly bonded to the replaced carbon must be deleted. xe2x80x9cHeteroalkylenexe2x80x9d is an alkylene group (as defined herein) wherein at least one of the carbon atoms is replaced with a heteroatom. Preferred heteroatoms are nitrogen, oxygen, sulfur, and halogen. A heteroatom may, but typically does not, have the same number of valence sites as carbon. Accordingly, when a carbon is replaced with a heteroatom, the number of hydrogens bonded to the heteroatom may need to be increased or decreased to match the number of valence sites of the heteroatom, as explained elsewhere herein.
xe2x80x9cHeteroaralkenylxe2x80x9d is another name for heteroarylalkenylene, wherein at least one of the open bonding sites of an alkenylene group is bonded to a heteroaryl group.
xe2x80x9cHeteroaralkylxe2x80x9d is another name for heteroarylalkylene, wherein at least one of the open bonding sites of an alkylene group is bonded to a heteroalkyl group.
xe2x80x9cHeteroarylxe2x80x9d is a monovalent aromatic ring system containing carbon and at least one heteroatom in the ring. The heteroaryl group may, in various embodiments, have one heteroatom, or 1-2 heteroatoms, or 1-3 heteroatoms, or 1-4 heteroatoms in the ring. Heteroaryl rings may be monocyclic or polycyclic, where the polycyclic ring may contained fused, spiro or bridged ring junctions. In one embodiment, the heteroaryl is selected from monocyclic and bicyclic. Monocyclic heteroaryl rings may contain from about 5 to about 10 member atoms (carbon and heteroatoms), preferably from 5-7, and most preferably from 5-6 member atoms in the ring. Bicyclic heteroaryl rings may contain from about 8-12 member atoms, or 9-10 member atoms in the ring. The heteroaryl ring may be unsubstituted or substituted. In one embodiment, the heteroaryl ring is unsubstituted. In another embodiment, the heteroaryl ring is substituted. Exemplary heteroaryl groups include benzofuran, benzothiophene, furan, imidazole, indole, isothiazole, oxazole, piperazine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, quinoline, thiazole and thiophene.
xe2x80x9cHeteroarylenexe2x80x9d is a polyvalent aromatic ring system containing carbon and at least one heteroatom in the ring. In other words, a heteroarylene group is a heteroaryl group that has more than one open site for bonding to other groups. The heteroarylene group may, in various embodiments, have one heteroatom, or 1-2 heteroatoms, or 1-3 heteroatoms, or 1-4 heteroatoms in the ring. Heteroarylene rings may be monocyclic or polycyclic, where the polycyclic ring may contained fused, spiro or bridged ring junctions. In one embodiment, the heteroaryl is selected from monocyclic and bicyclic. Monocyclic heteroarylene rings may contain from about 5 to about 10 member atoms (carbon and heteroatoms), preferably from 5-7, and most preferably from 5-6 member atoms in the ring. Bicyclic heteroarylene rings may contain from about 8-12 member atoms, or 9-10 member atoms in the ring.
xe2x80x9cHeteroatomxe2x80x9d is a halogen, nitrogen, oxygen, silicon or sulfur atom. Groups containing more than one heteroatom may contain different heteroatoms.
xe2x80x9cPharmaceutically acceptable saltxe2x80x9d and xe2x80x9csalts thereofxe2x80x9d in the compounds of the present invention refers to acid addition salts and base addition salts.
Acid addition salts refer to those salts formed from compounds of the present invention and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and/or organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
Base addition salts refer to those salts formed from compounds of the present invention and inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Suitable salts include the ammonium, potassium, sodium, calcium and magnesium salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaines, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, and the like.
In one aspect the present invention provides 1,2,3-thiadiazole compounds, where such compounds include the heterocyclic ring system of formula (A). Formula (A) also indicates the numbering system used to uniquely identify each atom of the ring. 
Accordingly, the present invention provides compounds that include a five-membered ring, with unsaturation between the two ring nitrogen and the two ring carbons, i.e., unsaturation between the atoms at positions 2 and 3, and between 4 and 5.
The present inventors have discovered that certain derivatives of the 1,2,3-thiadiazole ring system have desirable bioactivity that render them useful in pharmaceutical compositions and methods of treatment, etc. More specifically, in one aspect, the present invention provides compounds of formula (1) 
and stereoisomers, solvates, and pharmaceutically acceptable salts thereof.
In formula (1), each of R1, R2, R3 and R4 is independently selected from hydrogen, R5, R6, and R7. R5 is selected from alkyl, heteroalkyl, aryl and heteroaryl; R6 is selected from (R5)n-alkylene, (R5)n-heteroalkylene, (R5)n-arylene and (R5)n-heteroarylene; R7 is selected from (R6)n-alkylene, (R6)n-heteroalkylene, (R6)n-arylene, and (R6)n-heteroarylene; and n is selected from 0, 1, 2, 3, 4 and 5. In formula (1), R1 and R2 may together form a heterocyclic structure including the nitrogen to which they are both attached, and R3 and R4 may together form a heterocyclic structure including the nitrogen to which they are both attached. Also in formula (1), each of L1 and L2 is independently selected from xe2x80x94A1xe2x80x94A2xe2x80x94A3xe2x80x94 where each of A1, A2, and A3 is independently selected from a direct bond, alkylene, heteroalkylene, arylene and heteroarylene.
In one aspect, each of R1, R2, R3 and R4 is a C1-C20 group selected from alkyl (e.g., alkyl and cycloalkyl, such as ethyl, propyl, butyl, hexyl, cyclohexyl, and adamantyl), heteroalkyl (e.g., CH3CH2xe2x80x94O-carbonyl, furanyl-carbonyl, hexyl-carbonyl, and adamantyl-carbonyl), aryl (e.g., phenyl and naphthyl), and heteroaryl (e.g., pyridyl). In another aspect, each of R1, R2, R3 and R4 is additionally, or alternatively, selected from alkylarylene (e.g., methylphenyl, ethylphenyl and cyclohexylphenyl), heteroalkylarylene (e.g., bromophenyl and methoxyphenyl), alkylheteroarylene (e.g., methylpyridyl), heteroalkylheteroarylene (e.g., methoxypyridyl), arylalkylene (e.g., phenylmethylene (i.e., benzyl) and phenylethylene), heteroarylalkylene (e.g., pyridyl-CH2xe2x80x94), arylheteroalkylene (e.g., phenylcarbonyl (ie., benzoyl), naphthylcarbonyl, and phenyl-CH2CH2-carbonyl), heteroarylheteroalkylene (e.g., pyridyl-carbonyl), arylarylene (e.g., biphenyl), heteroarylarylene (e.g., pyridyl-phenyl), heteroarylheteroarylene (e.g., pyridyl-pyridyl), and arylheteroarylene (e.g., phenyl-pyridyl).
In one aspect, R1, R2, R3, and R4 are each independently selected from hydrogen, alkyl, heteroalkyl, aryl, heteroaryl, heterocycloalkyl, cycloalkylalkylene, arylalkylene, heteroarylalkylene, heterocycloalkylalkylene; alkyl-O, heteroalkyl-O, aryl-O, heteroaryl-O, cycloalkyl-O, heterocycloalkyl-O, cycloalkylalkylene-O, arylalkylene-O, heteroarylalkylene-O, heterocycloalkylalkylene-O; alkyl-CO, heteroalkyl-CO, aryl-CO, heteroaryl-CO, cycloalkyl-CO, heterocycloalkyl-CO, cycloalkylalkylene-CO, arylalkylene-CO, heteroarylalkylene-CO, heterocycloalkylalkylene-CO; alkyl-CONH, heteroalkyl-CONH, aryl-CONH, heteroaryl-CONH, cycloalkyl-CONH, heterocycloalkyl-CONH, cycloalkylalkylene-CONH, arylalkylene-CONH, heteroarylalkylene-CONH, heterocycloalkylalkylene-CONH; alkyl-OCO, heteroalkyl-OCO, aryl-OCO, heteroaryl-OCO, cycloalkyl-OCO, heterocycloalkyl-OCO, cycloalkylalkylene-OCO, arylalkylene-OCO, heteroarylalkylene-OCO, heterocycloalkylalkylene-OCO; alkyl-SO2, heteroalkyl-SO2, aryl-SO2, heteroaryl-SO2, cycloalkyl-SO2, heterocycloalkyl-SO2, cycloalkylalkylene-SO2, arylalkylene-SO2, heteroarylalkylene-SO2, and heterocycloalkylalkylene-SO2.
In one aspect, R1, R2, R3, and R4 are each independently selected from hydrogen, alkyl, heteroalkyl, aryl, heteroaryl, heterocycloalkyl, heteroalkyl. In one aspect, R1 and R2 are each hydrogen.
In one aspect, R3 is hydrogen or alkyl. In another aspect, R3 is hydrogen.
In one aspect, R4 is R6, where R6 is selected from (R5)n-alkylene, (R5)n-heteroalkylene, (R5)n-arylene and (R5)n-heteroarylene, and R5 is selected from alkyl, heteroalkyl, aryl and heteroaryl, and n is selected from 0, 1, 2, 3, 4 and 5. In another aspect, R4 is R6, where R6 is selected from (R5)n-arylene and (R5)n-heteroarylene, and R5 is selected from alkyl and heteroalkyl, and n is selected from 0, 1, 2, 3, 4 and 5. In another aspect, R4 is R6, where R6 is selected from (R5)n-arylene and R5 is selected from alkyl and heteroalkyl, and n is selected from 0, 1, 2, 3, 4 and 5.
In one aspect, the present invention provides compounds of formula (1), wherein each of L1 and L2 is independently selected from xe2x80x94A1xe2x80x94A2xe2x80x94A3xe2x80x94 such that each of A1 and A2 is a direct bond, and A3 is selected from a direct bond, alkylene, heteroalkylene, arylene and heteroarylene. In another aspect, the present invention provides compounds of formula (1), wherein each of L1 and L2 is independently selected from xe2x80x94A1xe2x80x94A2xe2x80x94A3xe2x80x94 such that each of A1 and A2 is a direct bond, and A3 is selected from a direct bond, alkyl, and heteroalkyl. In another aspect, the present invention provides compounds of formula (1), wherein each of L1 and L2 is independently selected from xe2x80x94A1xe2x80x94A2xe2x80x94A3xe2x80x94 such that each of A1, A2, and A3 is a direct bond. In another aspect, the present invention provides compounds that exclude carbonyl or thiocarbonyl as L1 or L2. In another aspect, L1 is carbonyl or thiocarbonyl while L2 is a direct bond. In another aspect, L1 is a direct bond while L2 is selected from carbonyl and thiocarbonyl.
In various aspects, the compounds of formula (1) exclude L1xe2x95x90direct bond, and/or L2xe2x95x90direct bond, and/or R1xe2x95x90H, and/or R2xe2x95x90carboxamide (i.e., H2Nxe2x80x94(Cxe2x95x90O)xe2x80x94), and/or R3xe2x95x90H, and/or R4xe2x95x90phenyl (i.e., C6H5).
Certain compounds encompassed by formula (1) have been described in the chemical literature, where these compounds were, for example, used as intermediates in various synthetic schemes, and/or studied for their chemical or physical properties, but were not recognized or reported to have any biological activity. Specific literature citations of this type include: Tarasov et al., Khim. Geterotsikl. Soedin. 8:1124-1127, 1996; Morzherin, Tarasov and Bakulev, Khim. Geterotsikl Soedin. 4:554-559, 1994; Morzherin, Bakulev, Dankova and Mokrushin, Khim. Geterotsikl Soedin 4:548-553, 1994; Shafran, Bakulev, Shevirin, and Kolobov, Khim. Geterotsikl. Soedin. 6:840-6, 1993; Dankova, Bakulev, and Morzherin, Khim. Geterotsikl. Soedin. 8:1106-1112, 1992; Bakulev, Lebedev, Dankova, Mokrushin, and Petrosyan, Tetrahedron 45(23):7329-7340, 1989; Kankova, Bakulev, Kolobov, Andosova, and Mokrushin, Khim. Geterotsikl, Soedin. 6:827-829, 1989; Dankova, Bakulev, Kolobov, Shishkina, Yasman, and Lebedev, Khim. Geterotsikl. Soedin 9:1269-1273, 1988; Bakulev, Kolobov, Grishakov, and Mokrushin, Izv. Akad. Nauk SSR, Ser. Khim. 1:193-195, 1988; Kolobov, Bakulev, Mokrushin, and Lebedev, Khim. Geterotsikl. Soedin 11:1503-1508, 1987; Bakulev, Dankova, Mokrushin, Sidorov, and Lebedev, Khim. Geterotsikl. Soedin. 6:845-849, 1987; Kolobov, Bakulev, and Mokrushin, Zh. Org. Khim. 23(5):1120-1122, 1987; Lebedev, Shevchenko, Kazaryan, Bakulev, Shafran, Kolobov, and Prosyan, Khim. Geterotsikl. Soedin. 5:681-689, 1987; Shafran, Bakulev, Mokrushin, and Validuda, Khim. Geterotsikl. Soedin. 5:691-696, 1986; Dankova, Bakulev, Mokrushin, and Shafran, Khim. Geterotsikl. Soedin, 10:1429-1430, 1985; and Shafran, Bakulev, Mokrushin, and Pushkareva, Khim. Geterotsikl. Soedin. 12:1696-1697, 1982; and Gewald and Hain, J. Prakt Chem. 317(2):329-336,1975.
To the extent a compound of formula (1) is described, and the preparation thereof is enabled, in one or more of these scientific publications, then in one aspect of the present invention such a compound is excluded from the scope of compounds encompassed by formula (1).
However, in another aspect, the present invention provides an isolated compound of formula (1). That is, a compound of formula (1) that is not substantially contaminated with, or otherwise in contact with any other compound. Accordingly, the present invention provides compound of formula (1) in substantially pure form, i.e., in a purity of greater than about 95% by weight, preferably greater than about 98%, and more preferably greater than about 99% by weight. In one aspect, the impurity in contact with a compound of formula (1) is an organic chemical, e.g., an organic solvent. In another aspect, the impurity in contact with a compound of formula (1) is another compound of formula (1). Thus, in one aspect, the present invention provides a compound of formula (1) that is pure in that it is not in contact with another compound of formula (1).
In a related aspect, the present invention provides a compound of formula (1) in the form of an isolated stereoisomer, where the isolated stereoisomer preferably has greater biological efficacy than other stereoisomeric forms of the compound. Thus, in one aspect, the present invention provides an isolated stereoisomer that is in contact with other stereoisomeric forms to a minimal extent, such that the molar ratio of isolated stereosiomer to other isomeric forms is greater than 95:5, preferably greater than 98:2, and still more preferably 99:1.
In another aspect, the present invention provides a compound of formula (1) in the form of a pharmaceutically acceptable salt.
Particularly where the scientific literature has employed a compound of formula (1) merely as an intermediate in a synthetic procedure, or in purely scientific study, this literature may fail to provide the compound in the form that is desirably employed in preparing a pharmaceutical composition. Thus, in various aspects, the present invention provides compounds of formula (1) in a desirably high purity, or at least purified away from undesirable chemicals, and/or in the form of a pharmaceutically acceptable salt that is desirably employed in preparing a pharmaceutical composition according to the present invention.
U.S. Pat. Nos. 4,101,548 and 4,171,363 disclose quinazoline compounds, and in particular 2-piperazinyl-6,7-dimethoxyquinazolines compounds that include a 1,2,3-thiadiazole terminal group, as well as precursors thereto, as shown in Formula (II), where M1 is hydrogen, lower alkyl, NH2 or NHCO2M2 in which M2 is lower alkyl. These compounds are disclosed as having antihypertensive properties. 
In one aspect, the present invention excludes compounds of formula (II) from the scope of compounds encompassed by formula (1) of the present invention. In another aspect, the present invention excludes 1,2,3-thiadiazole compounds used as precursors to compounds of formula (II) as set forth in U.S. Pat. Nos. 4,171,363 and 4,171,363.
U.S. Pat. Nos. 3,787,434 and 3,874,873 disclose herbicidal compounds and compositions that include 1,2,3-thiadiazole-5-yl ureas of formula (III), where M3 is selected from oxygen- and nitrogen-containing groups as defined in these patents. 
In one aspect, the present invention excludes compounds of formula (III) from the scope of compounds encompassed by formula (1) of the present invention. In another aspect, the present invention excludes 1,2,3-thiadiazole compounds used as precursors to compounds of formula (III) as set forth in U.S. Pat. Nos. 3,787,434 and 3,874,873.
In one aspect the present invention provides compounds of formula (1) wherein L1 is selected from Cxe2x95x90O and Cxe2x95x90S, and each of R1 and R2 is hydrogen. In one aspect thereof, L2 is a direct bond, so that the present invention provides compounds of formula (2) 
and stereoisomers, solvates, and pharmaceutically acceptable salts thereof, wherein R3 and R4 are each independently selected from hydrogen, R5, R6, and R7. R5 is selected from alkyl, heteroalkyl, aryl and heteroaryl; R6 is selected from (R5)n-alkylene, (R5)n-heteroalkylene, (R5)n-arylene and (R5)n-heteroarylene; R7 is selected from (R6)n-alkylene, (R6)n-heteroalkylene, (R6)n-arylene, and (R6)n-heteroarylene; and n is selected from 0, 1, 2, 3, 4 and 5. In formula (2), R3 and R4 may together form a heterocyclic structure including the nitrogen to which they are. both attached.
In one aspect, the present invention provides compounds of formulae (1) and (2) wherein R3 and R4 are independently selected from hydrogen, alkyl (e.g., C1-C20 alkyl and cycloalkyl, such as ethyl, propyl, butyl, hexyl, cyclohexyl, and adamantyl), heteroalkyl (e.g., CH3CH2xe2x80x94O-carbonyl, furanyl-carbonyl, hexyl-carbonyl, and adamantyl-carbonyl), aryl (e.g., phenyl and naphthyl), heteroaryl (e.g., pyridyl), alkylarylene (e.g., methylphenyl, ethylphenyl and cyclohexylphenyl), heteroalkylarylene (e.g., bromophenyl and methoxyphenyl), alkylheteroarylene (e.g., methylpyridyl), heteroalkylheteroarylene (e.g., methoxypyridyl), arylalkylene (e.g., phenylmethylene (i e., benzyl) and phenylethylene), heteroarylalkylene (e.g., pyridyl-CH2xe2x80x94), arylheteroalkylene (e.g., phenylcarbonyl (i.e., benzoyl), naphthylcarbonyl, and phenyl-CH2CH2-carbonyl), heteroarylheteroalkylene (e.g., pyridyl-carbonyl), arylarylene (e.g., biphenyl), heteroarylarylene (e.g., pyridylphenyl), heteroarylheteroarylene (e.g., pyridyl-pyridyl), arylheteroarylene (e.g., phenyl-pyridyl), alkylaryleneheteroalkylene (e.g., t-butylphenyl-carbonyl), alkylarylenealkylene (e.g., 2-methylbenzyl, 4-methylbenzyl, 4-ethylbenzyl), heteroalkylaryleneheteroalkylene (e.g., methoxyphenyl-carbonyl-, nitrophenyl-carbonyl), and heteroalkylarylenealkylene (e.g., 4-hydroxybenzyl).
In one aspect, the present invention provides compounds of formulae (1) and (2) wherein R3 is H.
In one aspect, the present invention provides compounds of formulae (1) and (2) wherein R3 and R4 are selected from hydrogen and hydrocarbon groups.
In one aspect, the present invention provides compounds of formulae (1) and (2) wherein R4 is phenyl or substituted phenyl. In one aspect thereof, the present invention provides compounds of formula (3), 
and stereoisomers, solvates, and pharmaceutically acceptable salts thereof, wherein, independently at each occurrence,
G is selected from oxygen and sulfur;
R3 and R8 are selected from hydrogen R5, R6, and R7, where R5 is selected from alkyl, heteroalkyl, aryl and heteroaryl; R6 is selected from (R5)n-alkylene, (R5)n-heteroalkylene, (R5)n-arylene and (R5)n-heteroarylene; R7 is selected from (R6)n-alkylene, (R6)n-heteroalkylene, (R6)n-arylene, and (R6)n-heteroarylene; and n is selected from 0, 1, 2, 3, 4 and 5.
In one aspect, the present invention provides compounds of formula (3) wherein R3 is hydrogen, alkyl, or heteroalkyl. In another aspect, the present invention provides compounds of formula (3) wherein R3 is hydrogen or alkyl. In another aspect, the present invention provides compounds of formula (3) wherein R3 is hydrogen.
In one aspect, the present invention provides compounds of formula (3) wherein R8 is R5; R5 is selected from alkyl, heteroalkyl, aryl and heteroaryl; and n is selected from 0, 1, 2, 3, 4 and 5. In one aspect, the present invention provides compounds of formula (3) wherein R8 is R5, and R5 is selected from alkyl and heteroalkyl, and n is selected from 1, 2, 3, 4 and 5.
In one aspect, the present invention provides compounds of formula (4) 
In formula (4), each of R1, R3 and R4 is independently selected from hydrogen, R5, R6, and R7. R5 is selected from alkyl, heteroalkyl, aryl and and (R5)n-heteroaryl; R7 is selected from (R6)n-alkylene, (R6)n-heteroalkylene, (R6)n-arylene and (R5)n-heteroarylene; R7 is selected from (R6)n-alkylene, (R6)n-heteroalkylene, (R6)n-arylene, and (R6)n-heteroarylene; and n is selected from 0, 1, 2, 3, 4 and 5. In formula (1), R3 and R4 may together form a heterocyclic structure including the nitrogen to which they are both attached. A1so in formula (4), L2 is xe2x80x94A1xe2x80x94A2xe2x80x94A3xe2x80x94 where each of A1, A2, and A3 is independently selected from a direct bond, alkylene, heteroalkylene, arylene and heteroarylene.
In various aspects, the present invention provides compounds of formula (4) wherein R1 is an alkyl group, and/or L2 is a direct bond, and/or R3 is hydrogen, and/or R4 is a hydrocarbon group.
In general, the following three methods and their variations as reported in the literature may be used to prepare 1,2,3-thiadiazole compounds of the present invention. The method of Pechmann and Nold (Ber. 29:2588, 1896) is one of the earliest reported methods to synthesize 1,2,3-thiadiazoles. This method utilizes the reaction of diazomethane and phenyl isothiocyanate (or more broadly, thiocarbonyl compounds) and may be utilized to prepare 5-amine substituted 1,2,3-thiadiazoles and 4,5-disubstituted 1,2,3-thiadiazoles. The method of Wolff (Ann. Chemie, 325:129, 1902) is another long-standing method to synthesize 1,2,3-thiadiazoles. According to the Wolff method, 1,2,3-thiadiazoles are obtained by treating diazoketones with a thionating reagent. A modification of the Wolff method utilizes diazoacetonitriles and hydrogen sulfide. The method of Hurd and Mori (J. Am. Chem. Soc. 77:5359, 1955) provides a reaction between hydrozones and thionyl chloride to afford 1,2,3-thiadiazole compounds, and is a preferred approach to preparing compounds of the present invention.
More specifically, compounds of the present invention may be prepared by one or more of the following general synthetic methods. One general synthetic method is illustrated in Scheme 1. 
As shown in Scheme 1, a solution of t-BuOK (1 equivalent) in anhydrous THF was cooled in an ice water bath under a nitrogen atmosphere. To this solution was added an acetoacetamide compound of the formula R2R1NC(xe2x95x90O)CH2C(O)CH3 (1 equivalent) in anhydrous THF, followed by the slow (15 min.) addition of an isothiocyanate compound of the formula R4xe2x80x94NCS (1 equivalent) with stirring. Suitable acetoacetamide and isothiocyanate compounds are commercially available and/or have been described in the chemical literature. The ice bath was removed and the reaction mixture was brought back to room temperature and stirred for about 1 hour. Water was added to the mixture and the resulting solution was kept stirring at room temperature for about 1 h. A solution of 1 N HCl solution was added to adjust the pH of the solution to about 7. The precipitate obtained was collected and dried to provide the amidoamine compound of the formula R2R1NC(xe2x95x90O)CH2C(S)NHR4.
The amidoamine compound of the formula R2R1NC(xe2x95x90O)CH2C(S)NHR4 (1 equivalent) was dissolved in a solution of anhydrous ethanol and triethylamine (1 equivalent). To this solution was added a diazo transfer agent, e.g., p-tosyl azide (1.2 equivalent). The mixture was warmed up to about 45xc2x0 C. and stirred for about 30 minutes, with the formation of a significant amount of solid precipitate. The solid was collected, washed with water and dried to provide the 1,2,3-thiadiazole product shown in Scheme 1.
In Scheme 1 R4 is selected from hydrogen, R5, R6, and R7. R5 is selected from alkyl, heteroalkyl, aryl and heteroaryl; R6 is selected from (R5)n-alkylene, (R5)n-heteroalkylene, (R5)n-arylene and (R5)n-heteroarylene; R7 is selected from (R6)n-alkylene, (R6)n-heteroalkylene, (R6)n-arylene, and (R6)n-heteroarylene; and n is selected from 0, 1, 2, 3, 4 and 5.
In one embodiment of the methodology of Scheme 1, R4 is 
where R8 is selected from hydrogen R5, R6, and R7, where R5 is selected from alkyl, heteroalkyl, aryl and heteroaryl; R6 is selected from (R5)n-alkylene, (R5)n-heteroalkylene, (R5)n-arylene and (R5)n-heteroarylene; R7 is selected from (R6)n-alkylene, (R6)n-heteroalkylene, (R6)n-arylene, and (R6)n-heteroarylene; and n is selected from 0, 1, 2, 3, 4 and 5.
A1so in Scheme 1, each of R1 and R2 is independently selected from hydrogen, R5, R6, and R7. R5 is selected from alkyl, heteroalkyl, aryl and heteroaryl; R6 is selected from (R5)n-alkylene, (R5)n-heteroalkylene, (R5)n-arylene and (R5)n-heteroarylene; R7 is selected from (R6)n-alkylene, (R6)n-heteroalkylene, (R6)n-arylene, and (R6)n-heteroarylene; and n is selected from 0, 1, 2, 3, 4 and 5. In formula (1), R1 and R2 may together form a heterocyclic structure including the nitrogen to which they are both attached.
Another general method is illustrated in Schemes 2 and 3. 
As illustrated in Scheme 2, an xcex1-cyanoamide is treated with a diazo transfer agent, e.g., p-tosyl azide, to introduce an azide group to the carbon between the carbonyl and cyano groups, i.e., the xcex1-carbon. This azide compound is then treated with a thiolating agent, e.g., Lawson""s agent, in order to generate a 4-amido-5-amino-1,2,3-thiadiazole compound. As illustrated in Scheme 3, the 4-amido-5-amino-1,2,3-thiadiazole compound formed by Scheme 2 is a versatile intermediate in the preparation of 1 ,2,3-thiadiazole compounds of the present invention.
In Scheme 3, xe2x80x9cExe2x80x9d, xe2x80x9cE1xe2x80x9d and xe2x80x9cE2xe2x80x9d (collectively xe2x80x9cExe2x80x9d) each represent a group selected from hydrogen, R5, R6, and R7, where R5 is selected from alkyl, heteroalkyl, aryl and heteroaryl; R6 is selected from (R5)n-alkylene, (R5)n-heteroalkylene, (R5)n-aryiene and (R5)n-heteroarylene; R7 is selected from (R6)n-alkylene, (R6)n-heteroalkylene, (R6)n-arylene, and (R6)n-heteroarylene; and n is selected from 0, 1, 2, 3, 4 and 5. In one embodiment, E is selected from hydrogen, R5 and R6. In another embodiment, E is selected from hydrogen and R5. In one embodiment, E is a hydrocarbon group.
Another general method is illustrated in Schemes 4, 5 and 6. 
As illustrated in Scheme 4, an xcex1-cyanocarboxylic ester is treated with a diazo transfer agent, e.g., p-tosyl azide, to introduce an azide group to the carbon between the carbonyl and cyano groups. This azide compound is then treated with a thiolating agent, e.g., Lawson""s agent, in order to generate a 4-carboxyester-5-amino-1,2,3-thiadiazole compound. As illustrated in Schemes 5 and 6, the 4-amido-5-amino-1,2,3-thiadiazole compound formed by Scheme 4 is a versatile intermediate in the preparation of 1,2,3-thiadiazole compounds of the present invention. 
In Schemes 5 and 6, xe2x80x9cRxe2x80x9d, xe2x80x9cRxe2x80x2xe2x80x9d and xe2x80x9cRxe2x80x3xe2x80x9d (collectively xe2x80x9cRxe2x80x9d) each represent a group selected from hydrogen, R5, R6, and R7, where R5 is selected from alkyl, heteroalkyl, aryl and heteroaryl; R6 is selected from (R5)n-alkylene, (R5)n-heteroalkylene, (R5)n-arylene and (R5)n-heteroarylene; R7 is selected from (R6)n-alkylene, (R6)n-heteroalkylene, (R6)n-arylene, and (R6)n-heteroarylene; and n is selected from 0, 1, 2, 3, 4 and 5. In one embodiment, R is selected from hydrogen, R5 and R6. In another embodiment, R is selected from hydrogen and R5. In one embodiment, R is a hydrocarbon group. Another general synthetic methodology is illustrated in Scheme 7. 
As shown in Scheme 7, an acetate ester (CH3xe2x80x94CO2xe2x80x94R) is treated with base, e.g., lithium diisopropylamide (LDA) at reduced temperature (typically xe2x88x9278xc2x0 C.), followed by addition of an isothiocyanate (Rxe2x80x2xe2x80x94NCS) where Rxe2x80x2 represents hydrogen, R5, R6, and R7, where R5 is selected from alkyl heteroalkyl, aryl and heteroaryl; R6 is selected from (R5)n-alkylene, (R5)n-heteroalkylene, (R5)n-arylene and (R5)n-heteroarylene; R7 is selected from (R6)n-alkylene, (R6)n-heteroalkylene, (R6)n-arylene, and (R6)n-heteroarylene; and n is selected from 0, 1, 2, 3, 4 and 5. The product, Rxe2x80x2xe2x80x94NHxe2x80x94C(xe2x95x90S)xe2x80x94CH2xe2x80x94CO2xe2x80x94R, is treated with a diazo transfer agent, e.g., p-tosyl azide to yield a 5-carboxylic ester-1,2,3-thiadiazole, which may be hydrolyzed to provide the corresponding 5-carboxylic acid-1,2,3-thiadiazole.
In one embodiment, Rxe2x80x2 is selected from hydrogen, R5 and R6. In another embodiment, Rxe2x80x2 is selected from hydrogen and R5. In one embodiment, Rxe2x80x2 is a hydrocarbon group.
The compounds of this invention can be incorporated into a variety of formulations for therapeutic administration. More particularly, the compounds of the present invention can be formulated into pharmaceutical compositions by combination with appropriate pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. As such, administration of the compounds can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc., administration. The active agent may be systemic after administration or may be localized by the use of regional administration, intramural administration, or use of an implant that acts to retain the active dose at the site of implantation.
In pharmaceutical dosage forms, the compounds may be administered in the form of their pharmaceutically acceptable salts. They may also be used in appropriate association with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.
For oral preparations, the compounds can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
The compounds can be formulated into preparations for injections by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
The compounds can be utilized in aerosol formulation to be administered via inhalation. The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
Furthermore, the compounds can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa bufter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more compounds of the present invention. Similarly, unit dosage forms for injection or intravenous administration may comprise the compound of the present invention in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
Implants for sustained release formulations are well-known in the art. Implants are formulated as microspheres, slabs, etc. with biodegradable or non-biodegradable polymers. For example, polymers of lactic acid and/or glycolic acid form an erodible polymer that is well-tolerated by the host. The implant containing the inhibitory compounds is placed in proximity to the site of the tumor, so that the local concentration of active agent is increased relative to the rest of the body.
The term xe2x80x9cunit dosage formxe2x80x9d, as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
The combined use of the provided inhibitory compounds and other cytotoxic agents has the advantages that the required dosages for the individual drugs is lower, and the effect of the different drugs complementary. Depending on the patient and condition being treated and on the administration route, the subject inhibitory compounds may be administered in dosages of 0.1 xcexcg to 10 mg/kg body weight per day. The range is broad, since in general the efficacy of a therapeutic effect for different mammals varies widely with doses typically being 20, 30 or even 40 times smaller (per unit body weight) in man than in the rat. Similarly the mode of administration can have a large effect on dosage. Thus for example oral dosages in the rat may be ten times the injection dose. Higher doses may be used for localized routes of delivery.
A typical dosage may be a solution suitable for intravenous administration; a tablet taken from two to six times daily, or one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient, etc. The time-release effect may be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release.
Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Some of the specific compounds are more potent than others. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means. A preferred means is to measure the physiological potency of a given compound.
For use in the subject methods, the subject compounds may be formulated with other pharmaceutically active agents, particularly other anti-metastatic, anti-tumor or anti-angiogenic agents. Angiostatic compounds of interest include angiostatin, endostatin, carboxy terminal peptides of collagen alpha (XV), etc. Cytotoxic and cytostatic agents of interest include adriamycin, alkeran, Ara-C, BICNU, busulfan, CNNU, cisplatinum, cytoxan, daunorubicin, DTIC, 5-FU, hydrea, ifosfamide, methotrexate, mithramycin, mitomycin, mitoxantrone, nitrogen mustard, velban, vincristine, vinblastine, VP-16, carboplatinum, fludarabine, gemcitabine, idarubicin, irinotecan, leustatin, navelbine, taxol, taxotere, topotecan, etc.
The subject compounds are administered to a subject having a hyperproliferative disorders, e.g. to inhibit tumor growth, to inhibit angiogenesis, to decrease inflammation associated with a lymphoproliferative disorder, to inhibit graft rejection, or neurological damage due to tissue repair, etc. The present compounds are useful for prophylactic or therapeutic purposes. As used herein, the term xe2x80x9ctreatingxe2x80x9d is used to refer to both prevention of disease, and treatment of pre-existing conditions. The prevention of proliferation is accomplished by administration of the subject compounds prior to development of overt disease, e.g. to prevent the regrowth of tumors, prevent metastatic growth, diminish restenosis associated with cardiovascularsurgery, etc. A1ternatively the compounds are used to treat ongoing disease, by stabilizing or improving the clinical symptoms of the patient.
The host, or patient, may be from any mammalian species, e.g. primate sp., particularly humans; rodents, including mice, rats and hamsters; rabbits; equines, bovines, canines, felines; etc. Animal models are of interest for experimental investigations, providing a model for treatment of human disease.
The susceptibility of a particular cell to treatment with the subject compounds may be determined by in vitro testing. Typically a culture of the cell is combined with a subject compound at varying concentrations for a period of time sufficient to allow the active agents to induce cell death or inhibit migration, usually between about one h and one week. For in vitro testing, cultured cells from a biopsy sample may be used. The viable cells left after treatment are then counted.
The dose will vary depending on the specific compound utilized, specific disorder, patient status, etc. Typically a therapeutic dose will be sufficient to substantially decrease the undesirable cell population in the targeted tissue, while maintaining patient viability. Treatment will generally be continued until there is a substantial reduction, e.g. at least about 50%, decrease in the cell burden, and may be continued until there are essentially none of the undesirable cells detected in the body.
The compounds also find use in the specific inhibition of signaling pathway mediated by protein kinases. Protein kinases are involved in signaling pathways for such important cellular activities as responses to extracellular signals and cell cycle checkpoints. Inhibition of specific protein kinases provides a means of intervening in these signaling pathways, for example to block the effect of an extracellular signal, to release a cell from cell cycle checkpoint, etc. Defects in the activity of protein kinases are associated with a variety of pathological or clinical conditions, where there is a defect in signaling mediated by protein kinases. Such conditions include those associated with defects in cell cycle regulation or in response to extracellular signals, e.g. hyperglycemia and diabetes Type I and Type II, immunological disorders, e.g. autoimmune and immunodeficiency diseases; hyperproliferative disorders, which may include psoriasis, arthritis, inflammation, angiogenesis, endometriosis, scarring, cancer, etc.
The compounds of the present invention are active in inhibiting purified kinase proteins, i.e. there is a decrease in the phosphorylation of a specific substrate in the presence of the compound. A protein kinase of particular interest in integrin linked kinase (ILK). ILK is a serine threonine kinase. The DNA and predicted amino acid sequence may be accessed at Genbank, no. U40282, or as published in Hannigan et al. (1996) Nature 379:91-96. ILK regulates integrin extracellular activity (ECM interactions) from inside the cell via its direct interaction with the integrin subunit. Interfering with ILK activity allows the specific targeting of integrin function, while leaving other essential signaling pathways intact. Increased levels of cellular ILK activity short circuits the normal requirement for adhesion to extracellular membrane in regulating cell growth. Thus, inhibiting ILK activity may inhibit anchorage-independent cell growth.
It is also known that many cell types undergo apoptosis if the appropriate contacts with extracellular matrix proteins are not maintained (anoikis). The induction of apoptosis by the subject compounds in such cells predicts an association with the ILK signaling pathway.
The compounds of the present invention bind to protein kinases at a high affinity, and find use as affinity reagents for the isolation and/or purification of such kinases. Affinity chromatography is used as a method of separating and purifying protein kinases and phosphatases using the biochemical affinity of the enzyme for inhibitors that act on it. The compounds are coupled to a matrix or gel. Preferably a microsphere or matrix is used as the support. Such supports are known in the art and commercially available. The inhibitor coupled support is used to separate an enzyme that binds to the inhibitor from a complex mixture, e.g. a cell lysate, that may optionally be partially purified. The sample mixture is contacted with the inhibitor coupled support under conditions that minimize non-specific binding. Methods known in the art include columns, gels, capillaries, etc. The unbound compounds are washed free of the resin, and the bound proteins are then eluted in a suitable buffer.
The compounds of the invention may also be useful as reagents for studying signal transduction or any of the clinical disorders listed throughout this application.
There are many disorders associated with a dysregulation of cellular proliferation. The conditions of interest include, but are not limited to, the following conditions.
The subject methods are applied to the treatment of a variety of conditions where there is proliferation and/or migration of smooth muscle cells., and/or inflammatory cells into the intimal layer of a vessel, resulting in restricted blood flow through that vessel, i.e. neointimal occlusive lesions. Occlusive vascular conditions of interest include atherosclerosis, graft coronary vascular disease after transplantation, vein graft stenosis, peri-anastomatic prosthetic graft stenosis, restenosis after angioplasty or stent placement, and the like.
Diseases where there is hyperproliferation and tissue remodelling or repair of reproductive tissue, e.g. uterine, testicular and ovarian carcinomas, endometriosis, squamous and glandular epithelial carcinomas of the cervix, etc. are reduced in cell number by administration of the subject compounds
Tumor cells are characterized by uncontrolled growth, invasion to surrounding tissues, and metastatic spread to distant sites. Growth and expansion requires an ability not only to proliferate, but also to down-modulate cell death (apoptosis) and activate angiogenesis to produce a tumor neovasculature. Angiogenesis may be inhibited by affecting the cellular ability to interact with the extracellular environment and to migrate, which is an integrin-specific function, or by regulating apoptosis of the endothelial cells. Integrins function in cell-to-cell and cell-to-extracellular matrix (ECM) adhesive interactions and transduce signals from the ECM to the cell interior and vice versa. Since these properties implicate integrin involvement in cell migration, invasion, intra- and extra-vasation, and platelet interaction, a role for integrins in tumor growth and metastasis is obvious.
Tumors of interest for treatment include carcinomas, e.g. colon, duodenal, prostate, breast, melanoma, ductal, hepatic, pancreatic, renal, endometrial, stomach, dysplastic oral mucosa, polyposis, invasive oral cancer, non-small cell lung carcinoma, transitional and squamous cell urinary carcinoma etc.; neurological malignancies, e.g. neuroblastoma, gliomas, etc.; hematological malignancies, e.g. childhood acute leukaemia, non-Hodgkin""s lymphomas, chronic lymphocytic leukaemia, malignant cutaneous T-cells, mycosis fungoides, non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis, T-cell rich cutaneous lymphoid hyperplasia, bullous pemphigoid, discoid lupus erythematosus, lichen planus, etc.; and the like.
Some cancers of particular interest include breast cancers, which are primarily adenocarcinoma subtypes. Ductal carcinoma in situ is the most common type of noninvasive breast cancer. In DCIS, the malignant cells have not metastasized through the walls of the ducts into the fatty tissue of the breast. Infiltrating (or invasive) ductal carcinoma (IDC) has metastasized through the wall of the duct and invaded the fatty tissue of the breast. Infiltrating (or invasive) lobular carcinoma (ILC) is similar to IDC, in that it has the potential metastasize elsewhere in the body. About 10% to 15% of invasive breast cancers are invasive lobular carcinomas.
A1so of interest is non-small cell lung carcinoma. Non-small cell lung cancer (NSCLC) is made up of three general subtypes of lung cancer. Epidermoid carcinoma (also called squamous cell carcinoma) usually starts in one of the larger bronchial tubes and grows relatively slowly. The size of these tumors can range from very small to quite large. Adenocarcinoma starts growing near the outside surface of the lung and may vary in both size and growth rate. Some slowly growing adenocarcinomas are described as alveolar cell cancer. Large cell carcinoma starts near the surface of the lung, grows rapidly, and the growth is usually fairly large when diagnosed. Other less common forms of lung cancer are carcinoid, cylindroma, mucoepidermoid, and malignant mesothelioma.
Melanoma is a malignant tumor of melanocytes. A1though most melanomas arise in the skin, they also may arise from mucosal surfaces or at other sites to which neural crest cells migrate. Melanoma occurs predominantly in adults, and more than half of the cases arise in apparently normal areas of the skin. Prognosis is affected by clinical and histological factors and by anatomic location of the lesion. Thickness and/or level of invasion of the melanoma, mitotic index, tumor infiltrating lymphocytes, and ulceration or bleeding at the primary site affect the prognosis. Clinical staging is based on whether the tumor has spread to regional lymph nodes or distant sites. For disease clinically confined to the primary site, the greater the thickness and depth of local invasion of the melanoma, the higher the chance of lymph node metastases and the worse the prognosis. Melanoma can spread by local extension (through lymphatics) and/or by hematogenous routes to distant sites. Any organ may be involved by metastases, but lungs and liver are common sites.
Other hyperproliferative diseases of interest relate to epidermal hyperproliferation, tissue remodelling and repair. For example, the chronic skin inflammation of psoriasis is associated with hyperplastic epidermal keratinocytes as well as infiltrating mononuclear cells, including CD4+memory T cells, neutrophils and macrophages.
The proliferation of immune cells is associated with a number of autoimmune and lymphoproliferative disorders. Diseases of interest include multiple sclerosis, rheumatoid arthritis and insulin dependent diabetes mellitus. Evidence suggests that abnormalities in apoptosis play a part in the pathogenesis of systemic lupus erythematosus (SLE). Other lymphoproliferative conditions the inherited disorder of lymphocyte apoptosis, which is an autoimmune lymphoproliferative syndrome, as well as a number of leukemias and lymphomas. Symptoms of allergies to environmental and food agents, as well as inflammatory bowel disease, may also be alleviated by the compounds of the invention.